LTBP-2 Competes With Tropoelastin for Binding to fibulin-5 and Heparin, and Is a Negative Modulator of Elastinogenesis

Matrix Biol. 2014 Feb;34:114-23. doi: 10.1016/j.matbio.2013.10.007. Epub 2013 Oct 19.

Abstract

Latent transforming growth factor-beta-1 binding protein-2 (LTBP-2) is a protein of ill-defined function associated with elastic fibers during elastinogenesis. Although LTBP-2 binds fibrillin-1, fibulin-5, and heparin/heparan sulfate, molecules critical for normal elastic fiber assembly, it does not interact directly with elastin or its precursor, tropoelastin. We investigated the modulating effect of LTBP-2 on two key interactions of tropoelastin during elastinogenesis a) with fibulin-5 and b) with heparan sulfate (using heparin). Firstly, using solid phase assays we showed that LTBP-2 bound fibulin-5 (Kd=26.47±5.68 nM) with an affinity similar to that of the tropoelastin-fibulin-5 interaction (Kd=24.66±5.64 nM). Then using a competitive binding assay we showed that LTBP-2 inhibited the tropoelastin-fibulin-5 interaction in a dose dependent manner with almost complete inhibition obtained with 5-fold molar excess of LTBP-2. Interestingly, a fragment of LTBP-2 containing the fibulin-5 binding sequence only partially inhibited the tropoelasin-fibulin-5 interaction suggesting that LTBP-2 was directly blocking only the C-terminal tropoelastin binding site on fibulin-5 and indirectly blocking tropoelastin binding to the N-terminal region. In parallel experiments heparin was shown to have minor inhibitory effects on fibulin-5 interactions with tropoelastin and LTBP-2. However, LTBP-2 was shown to significantly inhibit the binding of heparin to tropoelastin with 50% inhibition achieved with 10 fold molar excess of LTBP-2. Confocal microscopy of fibroblast matrix showed strong co-distribution of LTBP-2 with fibulin-5 and fibrillin-1 and partial co-distribution with heparan sulfate proteoglycans, perlecan and syndecan-4. Also addition of exogenous LTBP-2 to ear cartilage chondrocyte cultures blocked elastinogenesis in a concentration-dependent manner. Overall the results indicate that LTBP-2 may have a negative regulatory role during elastic fiber assembly, perhaps in displacing elastin microassemblies from complexes with fibulin-5 and/or cell surface heparan sulfate proteoglycans.

Keywords: Fibulin-5; Heparan sulfate; Heparin; LTBP-2; Tropoelastin.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cells, Cultured
  • Elastic Tissue / metabolism
  • Extracellular Matrix Proteins / metabolism*
  • Fibrillin-1
  • Fibrillins
  • Fibroblasts / metabolism
  • Heparin / genetics*
  • Heparin / metabolism
  • Heparitin Sulfate / genetics
  • Heparitin Sulfate / metabolism*
  • Humans
  • Latent TGF-beta Binding Proteins / metabolism*
  • Mice
  • Microfilament Proteins / metabolism
  • Protein Binding
  • Tropoelastin / genetics
  • Tropoelastin / metabolism*

Substances

  • Extracellular Matrix Proteins
  • FBLN5 protein, human
  • FBN1 protein, human
  • Fbn1 protein, mouse
  • Fibrillin-1
  • Fibrillins
  • LTBP2 protein, human
  • Latent TGF-beta Binding Proteins
  • Microfilament Proteins
  • Tropoelastin
  • Heparin
  • Heparitin Sulfate